The incessant damage to DNA from endogenous sources (e.g., oxygen radicals) is counteracted mainly by the base excision DNA repair (BER) pathway. Considerable knowledge of this process has been obtained by in vitro approaches, and genetic studies support important roles for key proteins such as the Ape1 abasic endonuclease in mammalian cells. BER sub-pathways have been described that direct the replacement of a single-nucleotide, or of several nucleotides in long-patch (LP) BER. The latter is especially important for the oxidized abasic site 2-deoxyribonolactone, but there are probably other lesions requiring LP-BER. What is missing is a robust and precise approach to characterizing these pathways in living cells. Understanding the actual pathway distribution in intact cells will illuminate genetic stability mechanisms in different cell types and for different DNA lesions. We propose to develop a novel approach to this problem, by establishing an assay using mass-labeled nucleotides incorporated in plasmid substrates for transfection into mammalian cells. In this approach, the target lesion will have the 3' downstream (or surrounding) nucleotides labeled with 13C or 15N, and placed in a non-replicating plasmid vector for transfection into mammalian cells. Repair in vivo will replace heavy nucleotides, the extend of which can be determined by subsequent mass spectrometry after recovery of the DNA. These processes will be facilitated by adjacent restriction enzyme sites and the presence of biotinylated nucleotides for affinity purification. There are two specific aims: 1. A plasmid vector we previously used to demonstrate LP-BER of 2-deoxyribonolactone in vitro will be used as a platform for lesions that delineate the single-nucleotide (uracil) and LP-BER (the stable abasic analog tetrahydrofuran) pathways, inserted via synthetic oligonucleotides containing surrounding mass-labeled nucleotides. These substrates will be tested initially in extracts from normal and DNA polymerase beta- deficient cells, with the latter expected to display predominantly LP-BER. 2. The vectors will be transfected into normal and POLB-deficient cells to assess the in vivo contribution of the BER sub-pathways acting on these (and eventually other) DNA lesions.